Figure 1.

Generation of Myogenic Progenitors from hPSCs via ^VP64dCas9^VP64-Mediated Activation of Endogenous PAX7

(A) Schematic of hPSC myogenic differentiation with small molecules and lentiviral activation of PAX7.

(B) The lentiviral constructs used for the gRNA and inducible ^VP64dCas9^VP64 and PAX7 cDNA expression.

(C) Representative phase-contrast images showing morphological changes during the first 10 days of differentiation. Scale bar, 200 μm.

(D) RNA was harvested at day 0 and day 2 for qRT-PCR analysis of mesodermal markers. Results are expressed as fold change over day 0 (mean ± SEM, n = 3 independent replicates).

(E) Representative fluorescence-activated cell sorting (FACS) plot at day 14 when ^VP64dCas9^VP64-2a-mCherry⁺ cells were sorted for expansion.

(F) Representative immunostaining of PAX7 at 5 days post sort. Scale bar, 100 μm.

(G) Growth of purified myogenic progenitors derived from iPSC differentiation during post-sort expansion phase was monitored over 2 weeks. Fold-growth over 2 weeks was significantly greater in ^VP64dCas9^VP64-treated cells compared with PAX7 cDNA-treated cells. p values were determined by one-way ANOVA followed by Tukey's post hoc test (mean ± SEM, n = 3 independent replicates).